Previous studies on the relationship between insulin resistance and obesity have demonstrated the advantages of weight loss and the disadvantages of weight gain. It is possible, however, that the overnutrition which preceeds obesity may be more important than the obese state for the development of insulin resistance. We have examined the effect of overnutrition on the development of insulin resistance in 15 subjects with normal glucose tolerance. Insulin resistance in the periphery and the liver was assessed with the euglycemic clamp at 100 and 1800 MuU/ml insulin using 3H-glucose to measure hepatic glucose production. Changes in glucose storage and oxidation were measured in vivo using indirect calorimetry. The results of this study indicate the importance of acute overnutrition for the development of insulin resistance. In addition, it relates overnutrition induced decreases in glucose disposal rates to decreases in both in vivo non-oxidative glucose disposal rates determined by indirect calorimetry and muscle glycogen synthase activity measured in vitro. Insulin binding and glucose transport measured in adipocytes were not decreased by overnutrition. The results suggest that overnutrition, independent of increased body mass, might result in reduced insulin mediated glucose storage rates as observed in obese subjects and that a reduction in glycogen synthase activity could contribute to this mechanism.